Serotonin (5-HT)-producing neurons projecting from brainstem to spinal cord (SC) are implicated in gating touch and pain transmission and modulating physiological processes from cardiorespiratory control to thermoregulation. Despite such vital and clinically significant roles, the specific serotonergic cells and SC circuits involved are poorly understood, challenged by the formidable anatomy and lack of molecular genetic tools of suitable specificity and resolving capacity. We have used intersectional genetic lineage tracing and transcriptomics genome-wide to reveal subtypes of 5-HT neurons, now providing sought-after means to map and functionally define the specific brainstem-SC circuits responsible for touch, pain, and homeostatic modulation. We propose a model in which transcriptionally and molecularly distinct 5-HT subtypes form unique projection patterns, selectively innervating regions of the spinal cord and modulating distinguishing sets of downstream cellular partners, creating circuits, and thus, functions, unique to each 5-HT neuron subtype. Intersectional labeling of 5-HT neuron subsets (Pet1+) identified by co-expression of Tachykinin1 (Tac1-Pet1 neurons) or Early growth response 2 (Egr2-Pet1 neurons) provides genetic access to two major caudal 5-HT neuron subtypes for visualizing their precise innervation patterns along rostrocaudal and dorsoventral spinal cord axes?previously unexplored. Further, because selective co-expression of neuromodulators distinguishes different descending 5-HT neuron subtypes, I will explore the neurochemical composition of their axonal terminals within the SC. To follow, I will use recently generated trans-synaptic tracing tools to visualize post- synaptic partners of 5-HT neuron subtypes and combine intersectional optogenetic tools with acute SC slice electrophysiology to probe the functional synaptic connectivity of Egr2-Pet1 and Tac1-Pet1 neuron descending projections. Results will inform how genetically-defined 5-HT neuron subtypes are organized functionally in the modulation of spinal cord circuits involved in sensory transmission or autonomic regulation. Deliverables may include selective therapeutic substrates to treat pain, hyperesthesia, and cardiorespiratory impairment.